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Active Gasdermin D Forms Plasma Membrane Pores and Disrupts Intracellular Compartments to Execute Pyroptotic Death in Macrophages During Canonical Inflammasome Activation

Russo, Hana
http://rave.ohiolink.edu/etdc/view?acc_num=case1499021688711516

Abstract Details

2017, Doctor of Philosophy, Case Western Reserve University, Pathology.
Pyroptosis is a regulated mode of lytic inflammatory cell death that promotes anti-microbial host defense but may contribute to sepsis. Pyroptosis requires canonical inflammasome assembly to mediate caspase-1 activation. Active caspase-1 cleaves gasdermin D (Gsdmd) to relieve an autoinhibitory interaction between the N and C-termini enabling N-terminal Gsdmd (N-Gsdmd) to oligomerize, insert into the plasma membrane (PM) as lytic pores, and execute pyroptotic cell death. To further characterize N-Gsdmd-dependent changes in plasma membrane (PM) permeability, we assayed propidium2+ (Pro2+) influx kinetics during NLRP3/Pyrin inflammasome activation in murine bone marrow-derived macrophages (BMDM). BMDM were characterized by rapid Pro2+ influx after initiation of NLRP3/Pyrin inflammasomes by nigericin (NG) or C. difficile toxin B (TcdB), respectively. No Pro2+ uptake in response to NG or TcdB was observed in Caspase-1-/- or ASC-/- BMDM. The cytoprotectant glycine profoundly suppressed NG and TcdB-induced lysis but not Pro2+ influx. The absence of Gsdmd expression resulted in suppression of NG-stimulated Pro2+ influx and pyroptotic lysis. Extracellular La3+ and Gd3+ reversibly blocked the induced Pro2+ influx and markedly delayed pyroptotic lysis without limiting upstream inflammasome activation. These caspase-1-induced pre-lytic, N-Gsdmd PM pores also facilitated the efflux of cytosolic ATP and influx of extracellular Ca2+. Whether N-Gsdmd also partitions into membranes of intracellular organelles to facilitate pyroptotic cell death signaling remains undefined. We stimulated WT, Gsdmd-/-, and Nlrp3-/- immortalized macrophages (iBMDMs) with NG, and also used La3+ as an inhibitor of PM pyroptotic pore activity to investigate the contribution of intracellular PM pore-independent perturbations to active Gsdmd-mediated cell death. The absence of Gsdmd attenuated NG-induced decreases in redox homeostasis. Inhibition of N-Gsdmd PM pore activity with La3+ uncovered a ROS-driven component of pyroptotic cell death that could be suppressed by the ROS scavenger N-acetylcysteine (NAC). NG perturbed mitochondrial respiration independently of Gsdmd and NLRP3, but mediated rapid lysosomal damage in a Gsdmd-dependent manner. These data suggest that in a setting of suppressed PM pyroptotic pore activity, NG stimulation induces an active Gsdmd-mediated and ROS-dependent death signaling cascade. These studies highlight the potentially extensive scope of N-Gsdmd-mediated cellular dysregulation, which should be considered when developing therapies that target Gsdmd-mediated pyroptosis.
George Dubyak (Advisor)
Alan Levine (Committee Chair)
Clifford Harding (Committee Member)
Pamela Wearsch (Committee Member)
Carlos Subauste (Committee Member)
Clive Hamlin (Committee Member)
189 p.
Cellular Biology; Immunology; Pathology
inflammasome; caspase-1; gasdermin D; Gsdmd; reactive oxygen species signaling; inflammatory cell death; lanthanide

Recommended Citations

Citations

  • Russo, H. (2017). Active Gasdermin D Forms Plasma Membrane Pores and Disrupts Intracellular Compartments to Execute Pyroptotic Death in Macrophages During Canonical Inflammasome Activation [Doctoral dissertation, Case Western Reserve University]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=case1499021688711516

    APA Style (7th edition)

  • Russo, Hana. Active Gasdermin D Forms Plasma Membrane Pores and Disrupts Intracellular Compartments to Execute Pyroptotic Death in Macrophages During Canonical Inflammasome Activation. 2017. Case Western Reserve University, Doctoral dissertation. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=case1499021688711516.

    MLA Style (8th edition)

  • Russo, Hana. "Active Gasdermin D Forms Plasma Membrane Pores and Disrupts Intracellular Compartments to Execute Pyroptotic Death in Macrophages During Canonical Inflammasome Activation." Doctoral dissertation, Case Western Reserve University, 2017. http://rave.ohiolink.edu/etdc/view?acc_num=case1499021688711516

    Chicago Manual of Style (17th edition)

case1499021688711516
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© 2017, all rights reserved.
This open access ETD is published by Case Western Reserve University School of Graduate Studies and OhioLINK.